bacteriological examination of water, milk and air

Bacteriological Examination of water,

milk and air

Water

Milk

Air

Examination of water, milk and air

Importance of water examination for pathogens

Water intended for human

consumption should not contain

any pathogenic organisms.

Water is used for many

applications either at home for

cooking ,washing or drinking

or in industries such as food

and pharmaceuticals.

It is also important for hospitals for example haemodialysis unit

Testing of water samples are done regularly to make sure of its safety

Supplies of drinking water contaminated with sewage may cause diseases such as: typhoid fever and cholera.

All sources of water should be tested regularly.

Microorganisms which indicate the fecal pollution in water are usually common intestinal commensal bacteria.

Most important indicators of fecal pollution of water

Escherichia coli: The essential indicator of fecal pollution of human

/animal origin. It is an important member of the coliform bacteria.

Coliforms are members of the enterobacteriaceae family and they

1. grow in the presence of bile salts.

2. produce acid and gas from fermentation of lactose at 37°C.

It is the commonly-used bacterial indicator of sanitary quality of food and water.

Enterococcus faecalis:• less numerous than E.coli in

human feces, but more resistant to chlorination.

Clostridium perfringens:• Less numerous in human feces• Its spores can survive in the

environment • Resist treatment processes than

most of the indicators.

Media used in bacteriological examination of water

1. For coliforms: MacConkey’s broth Containing bromocresol purple as the pH indicator.

To confirm the presence of E.coli : EMB agar + IMVC

Enterococcus faecalis: Glucose azide broth.

Clostridium perfringes: Differential reinforced clostridial medium.

Membrane Filtration Method

Determination of Most Probable Number (MPN) by dilution method

Pour plate technique

Methods Used in Bacteriological

Examination of Water

• Using Millipore Filter Apparatus

Membrane Filtration Method

MacConkey’s agar

Determination of MPN of Coliforms by Dilution Method

Water Sample

50 mlDSMB

5 x 10 mlDSMB

5 x 5 mlSSMB

50 ml watersample 10 ml water

sample1 ml water

sample

Results:

Positive tubes: showing production of acid or gas.

Acid production: change color of tube from purple to yellow

Gas production: detected in the Durham’s tube.

Purple Yellow

Gas

Determine no. of coliforms per 100 ml water sample (MPN) using the standard probability table.

1 3 2

MPN = 14

i.e: No. of coliform bacilli per 100 ml water sample is 14 cells.

Most probable number of coliforms by McCrady’s table

Using 10 fold serial dilution method

Viable Bacterial Count

9 ml Saline1 2 3Water sample

1 ml water

1 ml 1 ml

1/101/10 x 1/10

1/1001/100 x 1/10

1/1000

1 ml 1 ml 1 mlMelted NA

1 2 3

Results:

Dilution

factor 1 2 3 XX . y

10 x1 X1.y1

102 x2 X2.y2

103

x3 X3.y3

No. of colonies per plateY

No. of cells per 1 ml = X1.y1 + X2.y2 + X3.y3

3

Water

Milk

Air

Examination of water, milk and air

Human infections may be caused by

theingestion of animal milk which

contains microorganisms derived from:

a. Animal e.g. by contamination with its feces

b. The environment

c. Milk handlers such as dairy workers

Introduction:

Importance of milk examination for pathogens

It is important to examine milk for pathogens to ensure that it is safe to be consumed by man.

Milk is further used for

obtaining many milk products like cheese ,cream , butter and ice cream

E.coli Streptoccus pyogenesMycobacterium bovisBacillus anthracis Salmonella sp. Brucella sp.

Pathogenic bacteria present in milk

Determination of viable bacterial count:

Using the pour plate method after preparation of 10fold serial dilution from the milk sample with ringersolution.

Permissible number of bacterial flora in pasteurized milk is 5 x 104 cfu/ml

Permissible number of bacterial flora in long life milk is 10 cfu/ml

Methylene Blue Reduction Test

To determine quality of the milk Increasing the number of bacterial flora will reducethe color of methylene blue more rapidly due toincreasing consumption of oxygen.i.e.: The speed of reduction of methylene blue color isdirectly proportional to the number of bacteria presentin milk sample.

Methylene Blue Reduction Test

Results:

The shorter the decolorization time, the higherthe number of bacterial flora present in milk,and the poor quality of milk

Decolorization time Result

30 min – 2 hrs Poor quality 2 – 6 hrs fair quality 6 – 8 hrs good quality

Over 8 hrs excellent quality

Test for coliforms

Done by inoculation of MacConkey’s broth with 0.1 ml of milk sample.

Examine for the production of acid detected by changing the color of the medium from purple to yellow.

+ve result with gas production

-ve result

Water

Milk

Air

Examination of water, milk and air

Importance of keeping the micro- organisms count low in air

Surgical theaters

Food preparations

Drug materials

Cross infection and out breaks in hospitals

Number on bacteria in air depends on

Number of persons

Body movement

Disturbance of clothing

Methods of examination of air

a. Settle plate: Petri dishes containing an agar medium are left open

for a measured period of time. Large bacteria-carrying dust particles settle on the

medium. The plates are incubated and a count of the colonies is

formed

Blood agar is suitable for over all count

For detection of a particular microorganism suitable media is used .

Disadvantage of this method :Despite its simplicity it measures only therate of deposition of large particles fromthe air

b. Slit sampler It draws in air from the environment at a fixed rate and

causes the suspended particles to fall on the surface of the agar plate.

c. Air centrifuge

Centrifuging particles from the air on to a culture medium.

The sampled air passed along a tube lined with nutrient agar which was rotated on its long axis.

After sampling the strip is removed from the instrument and incubated then colonies can be counted.

Notice:

No level of contamination however low can be regarded as certainly safe.

Infection can be initiated by deposition of a single infected particle at a favorable site.

The probability of S. aureus initiated infection is low in comparison with Mycobacterium tuberculosis

Demonstration:

Air examination Settle plate Water examination Determination of MPN Milk examination Methylene blue reduction test